Using bran for softness in rye bread

ABSTRACT

A composition comprising: (a) a cereal bran comprising oat bran, rye bran or a mixture thereof; and (b) an enzyme composition comprising a cellulase enzyme, a glucanase enzyme, a xylanase enzyme or a mixture thereof; is disclosed. Methods of preparing the composition and its use in food compositions, especially its addition to flour compositions for making baked products such as bread, are also disclosed.

FIELD OF THE INVENTION

This invention relates to a composition containing a cereal bran. Italso relates to methods of producing the composition and its use in anumber of applications, in particular in food products and particularlyalthough not exclusively in baked products such as bread.

BACKGROUND TO THE INVENTION

Bran is the hard outer layers of cereal grain. It consists of thecombined aleurone and pericarp. Along with germ, it is an integral partof whole grains, and is often produced as a by-product of milling in theproduction of refined grains. Bran is particularly rich in dietary fibreand essential fatty acids and contains significant quantities of starch,protein, vitamins and dietary minerals. Bran is therefore often used toenrich food products, especially breads and other baked products, forthose aiming to increase their intake of dietary fibre.

Bran is largely insoluble in water. It is therefore desirable to atleast partially solubilise bran to make it easier to incorporate intofood compositions.

WO 2010/081869 and WO 2010/081870 both describe methods ofsolubilisation of cereal bran to produce a composition comprising atleast one part of the cereal bran that is solubilised. WO 2010/081870describes that this process is carried out by treating cereal bran withone or more cell-wall modifying enzyme; one or more starch modifyingenzyme; and optionally one or more further enzymes. WO 2010/081869describes that this process is carried out by treating cereal bran withone or more lipid modifying enzymes; and optionally one or more furtherenzymes such as xylanases and cellulases.

Both WO 2010/081869 and WO 2010/081870 describe that the solubilisedcereal bran may be incorporated into a flour composition which is thenused to form dough for baking. However, all of the examples of WO2010/081869 and WO 2010/081870 describe compositions and methods wherethe cereal bran is wheat bran. Neither WO 2010/081869 nor WO 2010/081870specifically disclose a composition including a cereal bran and anenzyme wherein the cereal bran is oat bran and/or rye bran and theenzyme is a cellulase enzyme, a glucanase enzyme and/or a xylanaseenzyme.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a composition comprising:

(a) a cereal bran comprising oat bran, rye bran or a mixture thereof;and(b) an enzyme composition comprising a cellulase enzyme, a glucanaseenzyme, a xylanase enzyme or a mixture thereof.

Typically, the composition is a liquid suspension (preferably an aqueoussuspension) containing the cereal bran and the enzyme composition.Typically, the enzyme composition causes the cereal bran to be at leastpartially modified and/or solubilised.

In another aspect, the invention provides a method of preparing theabove composition, comprising contacting the cereal bran with the enzymeand, optionally, other components of the composition. The method mayfurther comprise the addition of a liquid, typically water, to form anaqueous suspension of the cereal bran and the enzyme composition.

In a further aspect, the invention provides a flour compositioncomprising: (a) a flour; and (b) a composition as defined above.

In a yet further aspect, the invention provides a food productcontaining a composition as defined above or a flour composition asdefined above.

In a still further aspect, the invention provides a dough compositioncomprising: (a) the flour composition as defined above; and (b) water.

In a yet further aspect, the invention provides a baked productobtainable by baking the dough composition as defined above.

In a still further aspect, the invention provides a method ofsolubilising a cereal bran, said method comprising treating a liquidsuspension of said cereal bran with an enzyme; wherein:

(a) said cereal bran comprises oat bran, rye bran or a mixture thereof;and(b) said enzyme comprises a cellulase enzyme, a glucanase enzyme and/orxylanase enzyme or a mixture thereof.

In a yet further aspect, the invention provides solubilised cereal branobtained or obtainable according to the above method.

In a still further aspect, the invention provides a food ingredientobtained or obtainable by drying the above solubilised cereal bran.

In a yet further aspect, the invention provides use of a solubilisedcereal bran as defined above in the preparation of a food product.Typically, the food product is a dough or a baked product prepared fromdough.

In a still further aspect, the invention provides a kit of partscomprising:

(a) a cereal bran comprising oat bran, rye bran or a mixture thereof;(b) an enzyme comprising a cellulase enzyme, a glucanase and/or xylanaseenzyme or a mixture thereof;(c) instructions for use in a method as defined above; and(d) optionally other ingredients for a food product.

In a yet further aspect, the invention provides use of an enzyme toimprove the volume of bread containing a cereal bran, wherein:

(a) said cereal bran comprises oat bran, rye bran or a mixture thereof;and(b) said enzyme comprises a cellulase enzyme, a glucanase and/orxylanase enzyme or a mixture thereof.

In a still further aspect, the invention provides use of an enzyme toimprove the softness of bread containing a cereal bran, wherein:

(a) said cereal bran comprises oat bran, rye bran or a mixture thereof;and(b) said enzyme comprises a cellulase enzyme, a glucanase enzyme and/orxylanase enzyme or a mixture thereof.

Advantages and Surprising Findings

It has been surprisingly found by the present inventors that, when acereal bran comprising oat bran and/or rye bran is treated with anenzyme composition as defined herein, and the resulting compositionincorporated into a dough composition which is then baked to form bread,the resulting bread exhibits improved volume and improved softness. Thisis contrary to what would have been expected, as the prior art onlyspecifically discloses enzyme treated wheat bran compositions andnowhere discloses or suggests that a bread having sufficient softnessand volume could be produced using a flour to which oat bran or rye branhas been added.

In particular, it has been surprisingly found by the present inventorsthat, when a cereal bran comprising oat bran and/or rye bran is treatedwith an enzyme composition as defined herein, and the resultingcomposition incorporated into a dough composition including rye flourwhich is then baked to form bread, the resulting bread exhibits improvedvolume and softness. This is contrary to what would have been expected,as producing a bread of acceptable volume and softness using rye flourhas proved particularly difficult in the prior art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a picture showing the crumb of breads produced in the bakingtrial of Example 2; and

FIG. 2 is a picture showing the crumb of the breads produced in thebaking trial of Example 3.

GENERAL DEFINITIONS

Amino acids are referred to herein using the name of the amino acid, thethree letter abbreviation or the single letter abbreviation.

The term “protein”, as used herein, includes proteins, polypeptides, andpeptides. As used herein, the term “amino acid sequence” is synonymouswith the term “polypeptide” and/or the term “protein”. In someinstances, the term “amino acid sequence” is synonymous with the term“peptide”. In some instances, the term “amino acid sequence” issynonymous with the term “enzyme”.

The terms “protein” and “polypeptide” are used interchangeably herein.In the present disclosure and claims, the conventional one-letter andthree-letter codes for amino acid residues may be used. The 3-lettercode for amino acids as defined in conformity with the IUPAC IUB JointCommission on Biochemical Nomenclature (JCBN). It is also understoodthat a polypeptide may be coded for by more than one nucleotide sequencedue to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification.Before the exemplary embodiments are described in more detail, it is tounderstand that this disclosure is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present disclosure will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin this disclosure. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and eachrange where either, neither or both limits are included in the smallerranges is also encompassed within this disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anenzyme” includes a plurality of such candidate agents and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that such publicationsconstitute prior art to the claims appended hereto.

The term “cereal” as used herein refers to the fruits from a plant ofthe family Poaceae, such seed containing at least the bran comprisingthe aleurone, and the starchy endosperm, with or without the additionalpresence of pericarp, seed coat (alternatively called testa) and/orgerm. The term includes, but is not limited to species such as wheat,barley, oat, spelt, rye, sorghum, maize, and rice.

The term “solubilisation” as used herein refers to the solubilisation ofcereal bran in the methods according to the invention and is intended toinclude any degree of solubilisation. Accordingly the “solubilisation”may be to obtain 100% soluble material or it may be to obtain asolubilisation degree less than 100%, such as less than 70%, such as inthe range of 40%-60% or such as in the range of 20%-40%. In someembodiments the solubilisation degree is determined on dry matter versusdry matter bran.

Composition

In a first aspect, the invention provides a composition comprising:

(a) a cereal bran comprising oat bran, rye bran or a mixture thereof;and(b) an enzyme composition comprising a cellulase enzyme, a glucanaseenzyme, a xylanase enzyme or a mixture thereof.

This composition is also referred to herein as “the bran composition ofthe invention”.

Typically, the composition is a liquid suspension (preferably an aqueoussuspension) containing the cereal bran and the enzyme composition.

Bran

The composition of the invention comprises a cereal bran. The term“bran” as used herein refers to a cereal-derived milling fractionenriched in any or all of the tissues to be selected from aleurone,pericarp and seed coat, as compared to the corresponding intact seed.

The term “milling fraction”, as used herein, refers to all or part ofthe fractions resulting from mechanical reduction of the size of grains,through, as examples but not limited to, cutting, rolling, crushing,breakage or milling, with or without fractionation, through, as examplesbut not limited to, sieving, screening, sifting, blowing, aspirating,centrifugal sifting, windsifting, electrostatic separation, or electricfield separation.

In one embodiment, the cereal bran comprises, consists essentially of orconsists of oat bran. In one embodiment, the cereal bran comprises,consists essentially of or consists of rye bran.

The composition of the invention may additionally contain bran fromother grains in addition to the oat bran and/or rye bran. Examples ofother grains which may be acceptable include bran from wheat, barley,triticale, rice, and corn (maize).

In one embodiment the oat bran comprises at least 50%, such as at least60%, such as at least 70%, such as at least 80%, such as at least 90%,such as at least 95%, such as at least 97%, such as at least 98%, suchas at least 99%, such as at least 99.5%, by weight of the total weightof the bran in the composition.

In one embodiment the rye bran comprises at least 50%, such as at least60%, such as at least 70%, such as at least 80%, such as at least 90%,such as at least 95%, such as at least 97%, such as at least 98%, suchas at least 99%, such as at least 99.5%, by weight of the total weightof the bran in the composition.

When the cereal bran is contacted with the enzyme composition(particularly although not exclusively in an aqueous suspension), theactivity of the enzyme composition typically causes the cereal bran tobe at least partially modified. The activity of the enzyme compositionalso, or in the alternative, typically causes the cereal bran to be atleast partially solubilised. Without wishing to be bound by theory,these modifications are thought to assist in obtaining a bread productcontaining cereal bran (in particular, in which oat and/or rye bran hasbeen added to the flour used to form the dough) having acceptable andimproved volume and softness.

Following treatment with the enzyme composition, the suspension ofcereal bran may be dried to form a dried composition. Dried compositionsare typically more suitable for incorporating into flour compositions tobe made into dough to form baked products such as bread.

Enzyme Composition

The enzyme composition used in the present invention comprises acellulase enzyme, a glucanase enzyme, a xylanase enzyme or a mixture ofany thereof.

In one embodiment the enzyme composition used in the composition of thepresent invention comprises, consists essentially of or consists of acellulase enzyme. In one embodiment the enzyme composition used in thecomposition of the present invention comprises, consists essentially ofor consists of a glucanase enzyme. In one embodiment the enzymecomposition used in the composition of the present invention comprises,consists essentially of or consists of a xylanase enzyme. In oneembodiment the enzyme composition used in the composition of the presentinvention comprises, consists essentially of or consists of a mixture ofa cellulase enzyme and a glucanase enzyme. In one embodiment the enzymecomposition used in the composition of the present invention comprises,consists essentially of or consists of a mixture of a cellulase enzymeand a xylanase enzyme. In one embodiment the enzyme composition used inthe composition of the present invention comprises, consists essentiallyof or consists of a mixture of a glucanase enzyme and a xylanase enzyme.In one embodiment the enzyme composition used in the composition of thepresent invention comprises, consists essentially of or consists of amixture of a cellulase enzyme, a glucanase enzyme and a xylanase enzyme.

In one embodiment, the enzyme used in the present invention is aglucanase. Glucanases are enzymes that break down a glucan, which is apolysaccharide comprising (or consisting of) glucose sub-units. As theyperform hydrolysis of the glucosidic bond, they are hydrolases.

In one embodiment, the enzyme is an α-glucanase. The α-glucanase may bean α-1,4-glucanase (i.e. an enzyme that breaks down α-1,4-glucans), anα-1,6-glucanase, (i.e. an enzyme that breaks down α-1,6-glucan) or amixture thereof.

In one embodiment, the enzyme is a β-glucanase. The β-glucanase may be aβ-1,3-glucanase (i.e. an enzyme that breaks down β-1,3-glucans), aβ-1,4-glucanase (i.e. an enzyme that breaks down β-1,4-glucans), a β-1,6glucanase (i.e. an enzyme that breaks down β-1,6-glucans) or a mixtureof any thereof. Cellulase is a specific form of β-glucanase, whichperform the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,lichenin and cereal β-D-glucans.

In one embodiment, the enzyme is a mixture of α-glucanase and aβ-glucanase. In one embodiment, the enzyme is a glucanase other than acellulase. In one embodiment, the enzyme is a β-glucanase other than acellulase. In one embodiment, the enzyme is a β-1,4-glucanase other thana cellulase.

In some embodiments the enzyme composition may in addition to having aglucanase activity further comprise one or more of the activitiesselected from the group consisting of: a mannanase, a pectinase, axylanase, a glucuronidase, a galactanase. In one embodiment the enzymecomposition comprises glucanase (e.g. a β-glucanase) activity andmannanase activity. In one embodiment the enzyme composition comprisesendoglucanase (e.g. a β-glucanase) activity and pectinase activity.

In some embodiments the glucanase activity comprises at least 50%, suchas at least 60%, such as at least 70%, such as at least 80%, such as atleast 90%, such as at least 95%, such as at least 97%, such as at least98%, such as at least 99%, such as 100% of the total activity of theenzyme.

In some embodiments the glucanase enzyme is of plant origin. In someembodiments the glucanase enzyme is of bacterial origin. In someembodiments the glucanase enzyme is of fungal origin.

In one embodiment, the enzyme used in the present invention is acellulase. In this specification the term “cellulase” is understood asmeaning an enzyme that catalyzes cellulolysis (i.e. the decomposition ofcellulose and of some related polysaccharides); specifically, aβ-1,4-glucanase enzyme that performs the hydrolysis of1,4-beta-D-glucosidic linkages in cellulose, lichenin and cerealβ-D-glucans. Cellulases break down the cellulose molecule intomonosaccharides such as beta-glucose, or shorter polysaccharides andoligosaccharides.

The terms “cellulases” or “cellulolytic enzymes” as used herein areunderstood as comprising the cellobiohydrolases (EC 3.2.1.91), e.g.,cellobiohydrolase I and cellobiohydrolase II, as well as theendo-glucanases (EC 3.2.1.4) and beta-glucosidases (EC 3.2.1.21).

Included within the definition of cellulases are: endoglucanases (EC3.2.1.4) that cut the cellulose chains at random; cellobiohydrolases (EC3.2.1.91) which cleave cellobiosyl units from the cellulose chain endsand beta-glucosidases (EC 3.2.1.21) that convert cellobiose and solublecellodextrins into glucose. Among these three categories of enzymesinvolved in the biodegradation of cellulose, cellobiohydrolases are thekey enzymes for the degradation of native crystalline cellulose. Theterm “cellobiohydrolase I” is defined herein as a cellulose1,4-beta-cellobiosidase (also referred to as exo-glucanase,exo-cellobiohydrolase or 1,4-beta-cellobiohydrolase) activity, asdefined in the enzyme class EC 3.2.1.91, which catalyzes the hydrolysisof 1,4-beta-D-glucosidic linkages in cellulose and cellotetraose, by therelease of cellobiose from the non-reducing ends of the chains. Thedefinition of the term “cellobiohydrolase II activity” is identical,except that cellobiohydrolase II attacks from the reducing ends of thechains.

In some embodiments the cellulase enzyme is of plant origin. In someembodiments the cellulase enzyme is of bacterial origin. In someembodiments the cellulase enzyme is of fungal origin. In someembodiments the cellulase enzyme is derived from the fungus of the genusAspergillus. In some embodiments the cellulase enzyme is derived fromthe fungus of the genus Aspergillus niger. In some embodiments thecellulase enzyme is derived from the fungus of the species Trichodermareesei. In some embodiments the cellulase enzyme is derived from thefungus of the species Trichoderma reesei. In some embodiments thecellulase enzyme is derived from the fungus of the genus Humicola. Insome embodiments the cellulase enzyme is derived from the fungus of thespecies Humicola insolens.

In a preferred embodiment the cellulase enzyme used in the presentinvention is derived from the fungus of the species Trichoderma reesei.For example, the cellulase enzyme for use in the present invention maybe a crude or purified extract of a Trichoderma reseei fermentate. Aparticular example of such a composition is that described inPCT/EP2015/080439, unpublished at the filing date of the presentapplication. Such a composition is sold by DuPont under the name LaminexBG2.

In some embodiments the enzyme composition may in addition to having acellulase activity further comprise one or more of the activitiesselected from the group consisting of: a mannanase, a pectinase, axylanase, a glucuronidase, a galactanase. In some embodiments thecellulase activity comprises at least 50%, such as at least 60%, such asat least 70%, such as at least 80%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as 100% of the total activity of the enzyme.

In one embodiment the cellulase is an endo-glucanase (EC 3.2.1.4). Inone embodiment the cellulase is a cellobiohydrolase (EC 3.2.1.91). Inone embodiment the cellulase is a beta-glucosidase (EC 3.2.1.21).

The cellulases may comprise a carbohydrate-binding module (CBM) whichenhances the binding of the enzyme to a cellulose-containing fiber andincreases the efficacy of the catalytic active part of the enzyme. A CBMis defined as contiguous amino acid sequence within acarbohydrate-active enzyme with a discreet fold havingcarbohydrate-binding activity. For further information of CBMs see theCAZy internet server or Tomme et al. (1995) in Enzymatic Degradation ofInsoluble Polysaccharides (Saddler and Penner, eds.), Cellulose-bindingdomains: classification and properties, pp. 142-163, American ChemicalSociety, Washington. In a preferred embodiment the cellulolyticpreparation comprising a polypeptide having cellulolytic enhancingactivity (GH61A), preferably the one disclosed in WO 2005/074656.

In some embodiments the cellulase enzyme is a commercially availableproduct, such as GC220 available from Genencor, A Danisco Division, USor CELLUCLAST® 1.5L or CELLUZYME™ available from Novozymes A/S, Denmark.

Endoglucanases (EC No. 3.2.1.4) catalyses endo hydrolysis of1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (suchas carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin,beta-1,4 bonds in mixed beta-1,3 glucans such as cereal beta-D-glucansor xyloglucans and other plant material containing cellulosic parts. Theauthorized name is endo-1,4-beta-D-glucan 4-glucano hydrolase, but theabbreviated term endoglucanase is used in the present specification.Endoglucanase activity may be determined using carboxymethyl cellulose(CMC) hydrolysis according to the procedure of Ghose, Pure and Appl.Chem. 1987, 59, 257-268.

In some embodiments the endoglucanase is of plant origin. In someembodiments the endoglucanase is of fungal origin. In some embodimentsthe endoglucanase is of bacterial origin. In some embodimentsendoglucanases may be derived from a fungus of the genus Trichoderma,such as a fungus of the species Trichoderma reesei. In some embodimentsendoglucanases may be derived from a fungus of a strain of the genusHumicola, such as a fungus of the species Humicola insolens. In someembodiments endoglucanases may be derived from a fungus of the genusChrysosporium, such as a fungus of the species Chrysosporiumlucknowense.

The term “cellobiohydrolase” means a 1,4-beta-D-glucan cellobiohydrolase(E.C. 3.2.1.91), which catalyzes the hydrolysis of 1,4-beta-D-glucosidiclinkages in cellulose, cellooligosaccharides, or any beta-1,4-linkedglucose containing polymer, releasing cellobiose from the reducing ornon-reducing ends of the chain. Examples of cellobiohydroloses arementioned above including CBH I and CBH II from Trichoderma reesei;Humicola insolens and CBH II from Thielavia terrestris cellobiohydrolase(CELL6A). Cellobiohydrolase activity may be determined according to theprocedures described by Lever et al., 1972, Anal. Biochem. 47 273-279and by van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156; vanTilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288. The Lever etal. method is suitable for assessing hydrolysis of cellulose in cornstover and the method of van Tilbeurgh et al., is suitable fordetermining the cellobiohydrolase activity on a fluorescent disaccharidederivative.

The term “beta-glucosidase” means a beta-D-glucoside glucohydrolase(E.C. 3.2.1.21), which catalyzes the hydrolysis of terminal non-reducingbeta-D-glucose residues with the release of beta-D-glucose. For purposesof the present invention, beta-glucosidase activity is determinedaccording to the basic procedure described by Venturi et al., 2002, J.Basic Microbiol. 42: 55-66, except different conditions were employed asdescribed herein. One unit of beta-glucosidase activity is defined as1.0 μmol of p-nitrophenol produced per minute at 50° C., pH 5 from 4 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate, 0.01% TWEEN® 20.

In some embodiments the beta-glucosidase is of plant origin. In someembodiments the beta-glucosidase is of fungal origin. In someembodiments the beta-glucosidase is of bacterial origin. In oneembodiment the beta-glucosidase is derived from a fungus of the genusTrichoderma. In some embodiments the beta-glucosidase is a derived froma fungus of the species Trichoderma reesei, such as the beta-glucosidaseencoded by the bgl1 gene (see EP 562003). In one embodiment thebeta-glucosidase is derived from a fungus of the genus Aspergillus. Insome embodiments the beta-glucosidase is derived from Aspergillus oryzae(recombinantly produced in Aspergillus oryzae according to WO02/095014), Aspergillus fumigatus (recombinantly produced in Aspergillusoryzae according to Example 22 of WO 02/095014) or Aspergillus niger(1981, J. Appl. 3: 157-163). In one embodiment the beta-glucosidase isderived from a fungus of the genus Penicillium.

In one embodiment, the enzyme used in the present invention is axylanase. The term “xylanase” as used herein refers to an enzyme that isable to hydrolyze the beta-1,4 glycosyl bond in non-terminalbeta-D-xylopyranosyl-1,4-beta-D-xylopyranosyl units of xylan orarabinoxylan. Such enzymes may also be known as include 1,4-beta-D-xylanxylanohydrolase, 1,4-beta-xylan xylanohydrolase, beta-1,4-xylanxylanohydrolase, (1-4)-beta-xylan 4-xylanohydrolase,endo-1,4-beta-xylanase, endo-(1-4)-beta-xylanase,endo-beta-1,4-xylanase, endo-1,4-beta-D-xylanase, endo-1,4-xylanase,beta-1,4-xylanase, beta-xylanase, and beta-D-xylanase.

In some embodiments the enzyme composition may in addition to having axylanase activity further comprise one or more of the activitiesselected from the group consisting of: a mannanase, a pectinase, acellulase a glucuronidase, a galactanase. In some embodiments thexylanase activity comprises at least 50%, such as at least 60%, such asat least 70%, such as at least 80%, such as at least 90%, such as atleast 95%, such as at least 97%, such as at least 98%, such as at least99%, such as 100% of the total activity of the enzyme.

The xylanases can be derived from a variety of organisms. In oneembodiment the xylanase is of plant origin. In one embodiment thexylanase is of fungal origin. In one embodiment the xylanase is derivedfrom a fungus of the genus Aspergillus. In one embodiment the xylanaseis derived from a fungus of the genus Penicillium. In one embodiment thexylanase is derived from a fungus of the genus Disporotrichum. In oneembodiment the xylanase is derived from a fungus of the genusNeurospora. In one embodiment the xylanase is derived from a fungus ofthe genus Fusarium. In one embodiment the xylanase is derived from afungus of the genus Humicola. In one embodiment the xylanase is derivedfrom a fungus of the genus Trichoderma.

In one embodiment the xylanase is of bacterial origin. In one embodimentthe xylanase is derived from a bacterium of the genus Bacillus. In oneembodiment the xylanase is derived from a bacterium of the genusAeromonas. In one embodiment the xylanase is derived from a bacterium ofthe genus Streptomyces. In one embodiment the xylanase is derived from abacterium of the genus Nocardiopsis. In one embodiment the xylanase isderived from a bacterium of the genus Thermomyces. Examples of suitablexylanases are described in WO92/17573, WO92/01793, WO91/19782 andWO94/21785.

In one aspect of the invention, the xylanase used in the presentinvention is an enzyme classified as EC 3.2.1.8. The official name isendo-1,4-beta-xylanase. The systematic name is 1,4-beta-D-xylanxylanohydrolase. Other names may be used, such asendo-(1-4)-beta-xylanase; (1-4)-beta-xylan 4-xylanohydrolase;endo-1,4-xylanase; xylanase; beta-1,4-xylanase; endo-1,4-xylanase;endo-beta-1,4-xylanase; endo-1,4-beta-D-xylanase; 1,4-beta-xylanxylanohydrolase; beta-xylanase; beta-1,4-xylan xylanohydrolase;endo-1,4-beta-xylanase; beta-D-xylanase. The reaction catalyzed is theendohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.

In one aspect of the invention, the xylanase of the invention is axylanase of Glycoside Hydrolase (GH) Family 11. The term “of GlycosideHydrolase (GH) Family 11” means that the xylanase in question is or canbe classified in the GH family 11. In one aspect of the invention, thexylanase used according to the invention, is a xylanase having xylanaseactivity as measured in the “Xylanase assay” as described herein.

According to the Cazy(ModO) site, Family 11 glycoside hydrolases can becharacterised as follows:

Known Activities: xylanase (EC 3.2.1.8)

Mechanism: Retaining

Catalytic Nucleophile/Base: Glu (experimental)Catalytic Proton Donor: Glu (experimental)3D Structure Status: Fold: β-jelly roll

Clan: GH-C

As used herein, “Clan C” refers to groupings of families which share acommon three-dimensional fold and identical catalytic machinery (see,for example, Henrissat, B. and Bairoch, A., (1996) Biochem. J., 316,695-696).

As used herein, “Family 11” refers to a family of enzymes as establishedby Henrissat and Bairoch (1993) Biochem J., 293, 781-788 (see also,Henrissat and Davies (1997) Current Opinion in Structural Biol. 1997, 7,637-644). Common features for family 11 members include high genetichomology, a size of about 20 kDa and a double displacement catalyticmechanism (see Tenkanen et al., 1992; Wakarchuk et al., 1994). Thestructure of the family 11 xylanases includes two large β-sheets made ofβ-strands and α-helices.

Family 11 xylanases include the following: Aspergillus niger XynA,Aspergillus kawachii XynC, Aspergillus tubigensis XynA, Bacilluscirculans XynA, Bacillus punzilus XynA, Bacillus subtilis XynA,Neocalliniastix patriciarum XynA, Streptomyces lividans XynB,Streptomyces lividans XynC, Streptomyces thermoviolaceus XynII,Thermomonospora fusca XynA, Trichoderma harzianum Xyn, Trichodermareesei XynI, Trichoderma reesei XynII, Trichoderma viride Xyn.

In some embodiments the xylanase is derived from a filamentous fungus,preferably derived from a strain of Aspergillus, such as Aspergillusaculeatus; or a strain of Humicola, preferably Humicola lanuginosa. Thexylanase may preferably be an endo-1,4-beta-xylanase, more preferably anendo-1,4-beta-xylanase of GH 10 or GH11. Examples of commercialxylanases include Grindamyl H121 or Grindamyl Powerbake 930 from DuPontNutrition Biosciences ApS, Denmark or SHEARZYME™ and BIOFEED WHEAT™ fromNovozymes A/S, Denmark.

In some embodiments the enzyme composition also contains further enzymesin addition to the glucanase, cellulase and/or xylanase. Examples ofadditional enzymes which may be present include hydrolases (includingamylases), proteases lipases, phospholipases (e.g. a phospholipase A1 aphospholipase A2 a phospholipase B, a phospholipase C or a phospholipaseD) and glycolipases.

In one embodiment the enzyme composition contains a lipase. In oneembodiment the lipase is a triglyceridase. In one embodiment the lipaseis a glycolipases.

The lipase (e.g. a glycolipase) can be derived from a variety oforganisms. In one embodiment the lipase is of plant origin. In oneembodiment the lipase is of fungal origin. In one embodiment the lipaseis of bacterial origin.

In one embodiment the lipase is derived from a fungus of the genusFusarium. In one embodiment the lipase is derived from a fungus of thespecies Fusarium heterosporum.

In a particularly preferred embodiment, the lipase is the specificlipase derived from a fungus of the species Fusarium heterosporum,described generally and specifically in WO2005/087918.

Dosage

The one or more enzyme(s) for use in the present invention may be dosedas follows.

Typically, the cellulase enzyme is present in an amount of 0.1 mg to 100mg per 100 g bran. In one embodiment, the cellulase enzyme is present inan amount of 0.2 mg to 50 mg per 100 g bran. In one embodiment, thecellulase enzyme is present in an amount of 0.5 mg to 20 mg per 100 gbran. In one embodiment, the cellulase enzyme is present in an amount of1 mg to 10 mg per 100 g bran. These amounts are expressed as by weightof pure enzyme protein.

Typically, the xylanase enzyme is present in an amount of 0.006 mg to0.6 mg per 100 g bran. In one embodiment, the xylanase enzyme is presentin an amount of 0.012 mg to 0.3 mg per 100 g bran. In one embodiment,the xylanase enzyme is present in an amount of 0.03 mg to 0.12 mg per100 g bran. These amounts are expressed as by weight of pure enzymeprotein.

Typically, the cellulase enzyme is present in an amount of 30 to 3000units of cellulase activity per 100 g bran. In one embodiment, thecellulase enzyme is present in an amount of 60 to 1500 units ofcellulase activity per 100 g bran. In one embodiment, the cellulaseenzyme is present in an amount of 150 to 600 units of cellulase activityper 100 g bran. The Units of cellulase activity are measured inaccordance with the CMC-DNS activity assay described below.

Typically, the xylanase enzyme is present in an amount of 50 to 5000units of xylanase activity (XU) per 100 g bran. In one embodiment, thexylanase enzyme is present in an amount of 100 to 2500 units of xylanaseactivity (XU) per 100 g bran. In one embodiment, the xylanase enzyme ispresent in an amount of 250 to 1000 units of xylanase activity (XU) per100 g bran. The Units of xylanase activity are measured in accordancewith the activity assays described below.

Typically, the glucanase enzyme is present in an amount of 0.1 mg to 100mg per 100 g bran. In one embodiment, the glucanase enzyme is present inan amount of 0.2 mg to 50 mg per 100 g bran. In one embodiment, theglucanase enzyme is present in an amount of 0.5 mg to 20 mg per 100 gbran. In one embodiment, the glucanase enzyme is present in an amount of1 mg to 10 mg per 100 g bran. These amounts are expressed as by weightof pure enzyme protein.

Typically, the glucanase enzyme is present in an amount of 30 to 3000units of glucanase activity per 100 g bran. In one embodiment, theglucanase enzyme is present in an amount of 60 to 1500 units ofcellulase activity per 100 g bran. In one embodiment, the glucanaseenzyme is present in an amount of 150 to 600 units of cellulase activityper 100 g bran. The Units of cellulase activity are measured inaccordance with the CMC-DNS activity assay described below.

In a preferred embodiment the enzyme composition is a mixture of axylanase and a cellulase, the xylanase enzyme is present in an amount of0.006 mg to 0.6 mg per 100 g bran and the cellulase enzyme is present inan amount of 0.1 mg to 100 mg per 100 g bran. In a more preferredembodiment the enzyme composition is a mixture of a xylanase and acellulase, the xylanase enzyme is present in an amount of 0.03 mg to0.12 mg per 100 g bran and the cellulase enzyme is present in an amountof 1 mg to 10 mg per 100 g bran. These amounts are expressed as byweight of pure enzyme protein.

In a preferred embodiment the enzyme composition is a mixture of axylanase and a cellulase, the xylanase enzyme is present in an amount of50 to 5000 units of xylanase activity (XU) per 100 g bran and thecellulase enzyme is present in an amount of 30 to 3000 units ofcellulase activity per 100 g bran. In a more preferred embodiment theenzyme composition is a mixture of a xylanase and a cellulase, thexylanase enzyme is present in an amount of 250 to 1000 units of xylanaseactivity (XU) per 100 g bran and the cellulase enzyme is present in anamount of 150 to 600 units of cellulase activity per 100 g bran.

In an especially preferred embodiment, the enzyme composition is amixture of a bacterial xylanase (Grindamyl Powerbake 930 from DuPontNutrition Biosciences ApS) and a cellulase and/or glucanase enzyme whichis derived from a crude or purified extract of a Trichoderma reseeifermentate (as described in PCT/EP2015/080439, unpublished at the filingdate of the present application, and sold by DuPont under the nameLaminex BG2). Such an enzyme composition is also referred to in thisspecification as “TS-E 1732”. Preferably, in this mixture, the xylanaseenzyme is present in an amount of 0.006 mg to 0.6 mg per 100 g bran andthis cellulase enzyme is present in an amount of 0.1 mg to 100 mg per100 g bran. In a more preferred embodiment the enzyme composition is amixture of the specified xylanase and the specified cellulase, thespecified xylanase enzyme is present in an amount of 0.03 mg to 0.12 mgper 100 g bran and the specified cellulase enzyme is present in anamount of 1 mg to 10 mg per 100 g bran. These amounts are expressed asby weight of pure enzyme protein.

In an especially preferred embodiment, the enzyme composition is amixture of a bacterial xylanase (Grindamyl Powerbake 930 from DuPontNutrition Biosciences ApS) and a cellulase and/or glucanase enzyme whichis derived from a crude or purified extract of a Trichoderma reseeifermentate (as described in PCT/EP2015/080439, unpublished at the filingdate of the present application, and sold by DuPont under the nameLaminex BG2), the xylanase enzyme is present in an amount of 50 to 5000units of xylanase activity (XU) per 100 g bran and the cellulase enzymeis present in an amount of 30 to 3000 units of cellulase activity per100 g bran. In a more preferred embodiment the enzyme composition is amixture of the specified xylanase and the specified cellulase, thespecified xylanase enzyme is present in an amount of 250 to 1000 unitsof xylanase activity (XU) per 100 g bran and the specified cellulaseenzyme is present in an amount of 150 to 600 units of cellulase activityper 100 g bran.

Enzyme Activity Assays

The degree of solubilisation of the bran in the bran composition of thepresent invention may be measured according to the following method:

The degree of solubilisation of a plant material, e.g. cereal bran, canbe determined by suspending the insoluble plant material (typically 25%bran) in water with and without enzymes, incubate the suspension understirring and 50° C. for a controlled time (e.g. 30 to 1440 minutes).After solubilisation, the solubilised material is separated from theinsoluble material by centrifugation (20 min, 25000×g, room temp). Thedry matter content in the supernatant is determined either bylyophilizing part of the sample, or by a moisture analysis (Moistureanalyser, AND ML-50, Buch & Holm, Denmark). All the extraction buffercan not be recovered using this protocol, however, it is assumed thatthe concentration of soluble material is the same in the recoveredextraction buffer as in the not recovered extraction buffer, why acorrection is made for the extraction buffer used in total.

Having determined the dry matter content in the soluble fraction,knowing the amount of plant material taking into work and the amount ofextraction buffer, the solubilisation degree can be determined using thefollowing equation.

Solubilisation degree=(((gram dry matter/ml supernatant recovered)×(mlextraction buffer used))×100%)/gram plant material taken into work

Cellulase Activity Assay: By the CMC-DNS Procedure:

The assay of cellulase activity (e.g. endo-1,4-β-glucanase activity) isbased on the enzymatic hydrolysis of the 1,4-β-D-glucosidic bonds incarboxymethylcellulose (CM-Cellulose 4M, Megazyme Ltd) a β-1,4-glucan.The enzyme is diluted in double distilled water (ddH₂O) and 0.25 mlenzyme solution added to 1.75 ml substrate (1,5% CMC in 0.2M sodiumacetate buffer, pH 5.0) at 50° C. After 10 min of incubation a 2 ml 1%3,5-dinitrosalicylic acid (DNS) solution is added and the sample isplaced in boiling water bath for 5 min. The products of the reaction(β-1,4 glucan oligosaccharides) are determined colorimetrically at 540nm by measuring the resulting increase in reducing groups reacting withthe DNS. Enzyme activity is calculated from the relationship between theconcentration of reducing groups, as glucose equivalents, and absorbanceusing a glucose standard in the range 0.125-0.5 mg/ml. One unit ofcellulase activity is defined as the amount of enzyme which produces 1μmole glucose equivalents per minute under assay conditions.

Xylanase Assay (Endo-β-1,4-Xylanase Activity)

Samples were diluted in citric acid (0.1 M)-di-sodium-hydrogen phosphate(0.2 M) buffer, pH 5.0, to obtain approx. OD₅₉₀=0.7 in this assay. Threedifferent dilutions of the sample were pre-incubated for 5 minutes at40° C. At time=5 minutes, 1 Xylazyme tablet (crosslinked, dyed xylansubstrate, Megazyme, Bray, Ireland) was added to the enzyme solution ina reaction volume of 1 ml. At time=15 minutes the reaction wasterminated by adding 10 ml of 2% TRIS/NaOH, pH 12. Blanks were preparedusing 1000 μl buffer instead of enzyme solution. The reaction mixturewas centrifuged (1500×g, 10 minutes, 20° C.) and the OD of thesupernatant was measured at 590 nm. One xylanase unit (XU) is defined asthe xylanase activity increasing OD₅₉₀ with 0.025 per minute.

Host Cell

The host organism can be a prokaryotic or a eukaryotic organism. The atleast one enzyme may be obtainable (e.g. obtained) from any source. Theat least one enzyme may be a recombinant enzyme, for example an enzymethat is heterologous to the cell in which it is expressed. In otherembodiments the enzyme may be native to the cell in which it isexpressed. In one embodiment the one or more enzyme(s) is not obtainable(e.g. obtained) from a Trichoderma (e.g. Trichoderma reesei) host cell.

Alternative host cells may be fungi, yeasts or plants for example. Thehost cell may be any Bacillus cell other than B. subtilis. Preferably,said Bacillus host cell being from one of the following species:Bacillus licheniformis; B. alkalophilus; B. amyloliquefaciens; B.circulans; B. clausii; B. coagulans; B. firmus; B. lautus; B. lentus; B.megaterium; B. pumilus or B. stearothermophilus. Suitably the host cellmay a fungal host cell. Suitably the host cell may be any a Trichoderma,Meripilus, Humicola, Aspergillus, Fusarium or Chrysosporium host cell.Suitably, the host cell may be a protease deficient or protease minusstrain and/or an α-amylase deficient or α-amylase minus strain.

The term “heterologous”, as used herein, means a sequence derived from aseparate genetic source or species. A heterologous sequence is anon-host sequence, a modified sequence, a sequence from a different hostcell strain, or a homologous sequence from a different chromosomallocation of the host cell. As used herein, a “homologous” sequence is asequence that is found in the same genetic source or species i.e. it isnaturally occurring in the relevant species of host cell.

Regulatory Sequences

In some applications, an enzyme for use in the methods and/or usesdescribed herein may be obtained by operably linking a nucleotidesequence encoding same to a regulatory sequence which is capable ofproviding for the expression of the nucleotide sequence, such as by thechosen host cell (such as a B. licheniformis cell).

As used herein, the term “operably linked” refers to a juxtapositionwherein the components described are in a relationship permitting themto function in their intended manner. A regulatory sequence “operablylinked” to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with thecontrol sequences.

As used herein, the term “regulatory sequences” includes promoters andenhancers and other expression regulation signals.

As used herein, the term “promoter” is used in the normal sense of theart, e.g. an RNA polymerase binding site.

Enhanced expression of the nucleotide sequence encoding the enzymehaving the specific properties as defined herein may also be achieved bythe selection of regulatory regions, e.g. promoter, secretion leader andterminator regions that are not regulatory regions for the nucleotidesequence encoding the enzyme in nature. Suitably, the nucleotidesequence may be operably linked to at least a promoter.

Promoter

The promoter sequence to be used in accordance with the present methodsmay be heterologous or homologous to the sequence encoding any one ofthe enzymes for use in the present methods or uses described herein. Thepromoter sequence may be any promoter sequence capable of directingexpression of an enzyme in the host cell of choice.

Suitably, the promoter sequence may be homologous to a Bacillus species,for example B. licheniformis. Preferably, the promoter sequence ishomologous to the host cell of choice.

In another embodiment, the promoter may be homologous to a Geosmithiaspecies, for example Geosmithia emersonii.

Suitably, the promoter sequence may be homologous to the host cell.“Homologous to the host cell” means originating within the hostorganism; i.e. a promoter sequence which is found naturally in the hostorganism.

Suitably, the promoter sequence may be selected from the groupconsisting of a nucleotide sequence encoding: an α-amylase promoter, aprotease promoter, a subtilisin promoter, a glutamic acid-specificprotease promoter and a levansucrase promoter.

Suitably the promoter sequence may be a nucleotide sequence encoding:the LAT (e.g. the alpha-amylase promoter from B. licheniformis, alsoknown as AmyL), AprL (e.g. subtilisin Carlsberg promoter), EndoGluC(e.g. the glutamic-acid specific promoter from B. licheniformis), AmyQ(e.g. the alpha amylase promoter from B. amyloliquefaciens alpha-amylasepromoter) and SacB (e.g. the B. subtilis levansucrase promoter).

Other examples of promoters suitable for directing the transcription ofa nucleic acid sequence may include: the promoter of the Bacillus lentusalkaline protease gene (aprH); the promoter of the Bacillus subtilisalpha-amylase gene (amyE); the promoter of the Bacillusstearothermophilus maltogenic amylase gene (amyM); the promoter of theBacillus licheniformis penicillinase gene (penP); the promoters of theBacillus subtilis xylA and xylB genes; and/or the promoter of theBacillus thuringiensis subsp. tenebrionis CryIIIA gene.

Signal Peptide

The enzyme produced by a host cell by expression of the nucleotidesequence encoding the enzyme may be secreted or may be containedintracellularly depending on the sequence and/or the vector used. Asignal sequence may be used to direct secretion of the coding sequencesthrough a particular cell membrane. The signal sequences may be naturalor foreign to the coding sequence of the enzymes. For instance, thesignal peptide coding sequence may be obtained from an amylase orprotease gene from a Bacillus species, preferably from Bacilluslicheniformis.

Suitable signal peptide coding sequences may be obtained from one ormore of the following genes: maltogenic α-amylase gene, subtilisin gene,beta-lactamase gene, neutral protease gene, and/or prsA gene. In someembodiments, a nucleotide sequence encoding a signal peptide may beoperably linked to a nucleotide sequence encoding any one of the enzymesdisclosed herein. The enzyme for use in accordance with the presentmethods may be expressed in a host cell as defined herein as a fusionprotein.

Expression Vector

The term “expression vector” means a construct capable of in vivo or invitro expression. Preferably, the expression vector is incorporated inthe genome of the organism, such as a B. licheniformis host. The term“incorporated” preferably covers stable incorporation into the genome.

The nucleotide sequence encoding an enzyme as defined herein may bepresent in a vector, in which the nucleotide sequence is operably linkedto regulatory sequences such that the regulatory sequences are capableof providing the expression of the nucleotide sequence by a suitablehost organism (such as B. licheniformis), i.e. the vector is anexpression vector. The vectors may be transformed into a suitable hostcell as described above to provide for expression of a polypeptidehaving cellulase activity as defined herein. The choice of vector, e.g.plasmid, cosmid, virus or phage vector, genomic insert, will oftendepend on the host cell into which it is to be introduced. The presentmethods may cover other forms of expression vectors which serveequivalent functions and which are, or become, known in the art.

Once transformed into the host cell of choice, the vector may replicateand function independently of the host cell's genome, or may integrateinto the genome itself. The vectors may contain one or more selectablemarker genes—such as a gene which confers antibiotic resistance e.g.ampicillin, kanamycin, chloramphenicol or tetracycline resistance.Alternatively, the selection may be accomplished by co-transformation(as described in International Patent Application Publication No.WO91/17243). Vectors may be used in vitro, for example for theproduction of RNA or used to transfect or transform a host cell. Thevector may further comprise a nucleotide sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

Isolated

In one aspect, the enzyme is a recovered/isolated enzyme. Thus, one ormore of the enzyme(s) produced may be in an isolated form.

Purified

In one aspect, the enzyme(s) may be in a purified form. As used herein,the term “purified” means that the sequence is in a relatively purestate—e.g. at least about 51% pure, or at least about 75%, or at leastabout 80%, or at least about 90% pure, or at least about 95% pure or atleast about 98% pure.

Methods

The invention also provides a method for forming the composition of theinvention. The method comprises contacting the cereal bran with theenzyme composition and, optionally, other components of the composition.

The method is typically carried out by forming a liquid suspensioncontaining the cereal bran, the enzyme composition and a suspendingliquid, typically water.

Therefore, it is preferred that the method further comprises theaddition of water to form an aqueous suspension of the cereal bran andthe enzyme composition.

It has been found that the method of the invention works particularlywell when the liquid (preferably aqueous) suspension containing thecereal bran and the enzyme composition is heated to a temperature aboveroom temperature.

Therefore, in a preferred embodiment, the method according to theinvention further comprises heating the aqueous suspension.

Preferably the aqueous suspension is heated to a temperature of 30° C.to 100° C., such as 35° C. to 80° C., such as 40° C. to 60° C., such as45 to 55° C.

Preferably the aqueous suspension is heated for a time of 10 minutes to48 hours, such as 30 minutes to 24 hours, such as 1 to 12 hours, such as2 to 6 hours, such as 3 to 5 hours.

Prior to mixing the bran with the enzyme, in some embodiments of thepresent invention, the method may further comprising a step of i)fractionating the cereal grain to obtain endosperm, bran, and germ; ii)separating and distributing the endosperm, bran, and germ to allow themto be treated; and iii) milling the bran.

Typically, the method of the present invention causes the cereal bran tobe at least partially solubilised. Accordingly, in a further aspect, theinvention provides a method of solubilising a cereal bran, said methodcomprising treating a liquid suspension of said cereal bran with anenzyme; wherein:

(a) said cereal bran comprises oat bran, rye bran or a mixture thereof;and(b) said enzyme comprises a cellulase enzyme, a glucanase enzyme and/orxylanase enzyme or a mixture thereof.

In some embodiments, the degree of bran solubilisation is higher than20%, such as higher than 25%, such as higher than 30%, such as higherthan 35%, such as higher than 35%, such as higher than 40%, such ashigher than 50%, such as in the range of 40%-60%, such as in the rangeof 50%-60%. The degree of bran solubilisation is expressed as measuredin a “Dry matter content (%) in soluble fraction assay” and can bemeasured according to the procedure of Example 1 of WO2010/081870.

The method can be carried out on a liquid suspension with a low starchcontent. In some embodiments, less than 50%, such as less than 40%, suchas less than 30%, such as less than 20%, such as less than 10% by weightof the liquid suspension may be starch or components containing starch.

In some embodiments of the present invention, the method furthercomprises a step of drying the solubilised cereal bran obtained.

In some embodiments of the present invention, the method furthercomprises a step of spray drying the solubilised cereal bran obtained.

In some embodiments of the present invention, the method furthercomprises a step of lyophilisation of the solubilised cereal branobtained.

Applications and Food Compositions

The present invention further relates to the use of the bran compositionobtained according to the present invention. The bran composition isuseful as a food ingredient, particularly though not exclusively forincorporation into flour compositions for preparing dough and bakedproducts, such as bread, prepared from dough.

The composition of the present invention may be used as—or in thepreparation of—a food product. Here, the term “food product” is used ina broad sense—and covers food for humans as well as food for animals(i.e. a feed). In some aspects, the food is for human consumption. Thefood may be in the form of a solution or as a solid—depending on the useand/or the mode of application and/or the mode of administration.

In some embodiments of the present invention, the composition accordingto the invention is added directly in the production of the foodproduct.

In some embodiments of the present invention, the solubilised cerealbran obtained in the method according to the invention is added directlyas a mixture of soluble and insoluble cereal bran material in theproduction of the food product.

It is to be understood that the methods according to the presentinvention may produce an isolated solubilised fraction with only solublecereal bran material, such as when the soluble fraction is harvestedfrom a mixture of soluble and insoluble cereal bran material. In someembodiments such harvested soluble cereal bran material is used in theproduction of food products.

In other alternative embodiments, the solubilised cereal bran containingboth soluble and insoluble material may be used without furtherseparation or harvesting directly in production of food products.

In some embodiments of the present invention, the food product isselected from the group consisting of bread, a breakfast cereal, apasta, biscuits, cookies, snacks, and beer.

The composition of the present invention may also be used as a foodingredient. As used herein the term “food ingredient” includes aformulation which is or can be added to functional foods or foodstuffs,for example, as a nutritional supplement and/or fibre supplement. Theterm food ingredient as used here also refers to formulations which canbe used at low levels in a wide variety of products that requiregelling, texturising, stabilising, suspending, film-forming andstructuring, retention of juiciness and improved mouthfeel, withoutadding viscosity. The food ingredient may be in the form of a solutionor as a solid—depending on the use and/or the mode of application and/orthe mode of administration.

In one embodiment, the composition of the present invention may be—ormay be added to—food supplements. In one embodiment, the composition ofthe present invention may be—or may be added to—functional foods. Asused herein, the term “functional food” means food which is capable ofproviding not only a nutritional effect and/or a taste satisfaction, butis also capable of delivering a further beneficial effect to consumer.Accordingly, functional foods are ordinary foods that have components oringredients (such as those described herein) incorporated into them thatimpart to the food a specific functional—e.g. medical or physiologicalbenefit—other than a purely nutritional effect. Although there is nolegal definition of a functional food, most of the parties with aninterest in this area agree that they are foods marketed as havingspecific health effects.

Some functional foods are nutraceuticals. Here, the term “nutraceutical”means a food which is capable of providing not only a nutritional effectand/or a taste satisfaction, but is also capable of delivering atherapeutic (or other beneficial) effect to the consumer. Nutraceuticalscross the traditional dividing lines between foods and medicine.

Therefore, the invention further provides a food product containing acomposition according to the invention.

For certain aspects, the foodstuff is a bakery product—such as bread,Danish pastry, biscuits or cookies.

The bran composition of the invention may be incorporated into a flour.Therefore, in a further aspect, the invention provides a flourcomposition comprising:

(a) a flour; and(b) a composition according to the invention or a composition obtainableby a method according to the invention.

The flour may be made from any grain suitable for being milled to form aflour. Typical grains include wheat, barley, oat, rye and triticale,rice, and corn (maize). The flour may be made from the grain of onespecies or a mixture of species. In one embodiment, the flour comprises,consists essentially of or consists of wheat flour. In one embodiment,the flour comprises, consists essentially of or consists of rye flour.

In one aspect, the flour in the flour composition of the invention is awholegrain flour. In this aspect, the flour composition contains branand germ endogenous to the flour in addition to the bran from the brancomposition of the invention.

In one aspect, the flour in the flour composition of the invention is awhite flour (i.e. a flour from which the bran has been removed). In thisaspect, the bran present in the flour composition is solely from thebran composition of the invention.

In one embodiment, the flour comprises, consists essentially of orconsists of a mixture of wheat flour and rye flour. Typically, the flourcomprises, consists essentially of or consists of 20% to 40% by weight,such as 25 to 35%, by weight wheat flour and 60 to 80% by weight, suchas 65 to 75% by weight rye flour. Typically, the flour comprises,consists essentially of or consists of 30% by weight wheat flour and 70%by weight rye flour. Such a mixture is known to the person skilled inthe art as the flour composition for preparing “Mischbrot”.

In one embodiment, the bran composition according to the invention isadded to the flour. The composition can be mixed with the flour bymethods well known to those skilled in the art.

In one embodiment, the bran composition of the invention is present inthe flour composition of the invention in an amount of 0.1% to 20% byweight of the total weight of the flour composition. In one embodiment,the bran composition of the invention is present in the flourcomposition of the invention in an amount of 1% to 15% by weight of thetotal weight of the flour composition. In one embodiment, the brancomposition of the invention is present in the flour composition of theinvention in an amount of 2% to 10% by weight of the total weight of theflour composition. In one embodiment, the bran composition of theinvention is present in the flour composition of the invention in anamount of 5% to 7% by weight of the total weight of the flourcomposition. The amount by weight of the bran composition is expressedas bran dry solids, meaning that the stated amount of bran dry solidsforms the specified percentage of the total weight of the flourcomposition.

The flour composition of the invention can be used to form a doughcomposition by mixing with water. Accordingly, the invention alsoprovides a dough composition comprising: (a) the flour compositionaccording to the invention; and (b) water. In one embodiment the doughcomposition of the invention further comprises yeast.

The dough composition of the invention can be baked in order to preparebaked products, such as bread. Baking conditions are well known to theperson skilled in the art.

Accordingly, the invention also provides a baked product obtainable bybaking the dough composition of the invention. In one embodiment, saidbaked product comprises bread.

It has been surprisingly found according to the present invention thatwhen the bran composition of the invention is added to/incorporated intoa flour composition (particularly where the flour is wheat flour and/orrye flour), bread produced from the flour has a greater volume thanwould have been expected.

Accordingly, the invention further provides use of an enzyme to improvethe volume of bread containing a cereal bran, wherein:

(a) said cereal bran comprises oat bran, rye bran or a mixture thereof;and(b) said enzyme comprises a cellulase enzyme, a glucanase and/orxylanase enzyme or a mixture thereof.

It has been surprisingly found according to the present invention thatwhen the bran composition of the invention is added to/incorporated intoa flour composition (particularly where the flour is wheat flour and/orrye flour), bread produced from the flour is softer than would have beenexpected.

Accordingly, the invention further provides use of an enzyme to improvethe softness of bread containing a cereal bran, wherein:

(a) said cereal bran comprises oat bran, rye bran or a mixture thereof;and(b) said enzyme comprises a cellulase enzyme, a glucanase enzyme and/orxylanase enzyme or a mixture thereof.

In addition to bread and other baked products prepared from dough, thebran composition of the present invention can be used in the preparationof food products such as one or more of: jams, marmalades, jellies,dairy products (such as milk or cheese), meat products, poultryproducts, fish products and bakery products.

By way of example, the bran composition of the present invention can beused as ingredients to soft drinks, a fruit juice or a beveragecomprising whey protein, health teas, cocoa drinks, milk drinks andlactic acid bacteria drinks, yoghurt and drinking yoghurt, cheese, icecream, water ices and desserts, confectionery, biscuits cakes and cakemixes, snack foods, breakfast cereals, instant noodles and cup noodles,instant soups and cup soups, balanced foods and drinks, sweeteners,texture improved snack bars, fibre bars, bake stable fruit fillings,care glaze, chocolate bakery filling, cheese cake flavoured filling,fruit flavoured cake filling, cake and doughnut icing, heat stablebakery filling, instant bakery filling creams, filing for cookies,ready-to-use bakery filling, reduced calorie filling, adult nutritionalbeverage, acidified soy/juice beverage, aseptic/retorted chocolatedrink, bar mixes, beverage powders, calcium fortified soy/plain andchocolate milk, calcium fortified coffee beverage.

The bran composition of the present invention can further be used as aningredient in food products such as American cheese sauce, anti-cakingagent for grated & shredded cheese, chip dip, cream cheese, dry blendedwhip topping fat free sour cream, freeze/thaw dairy whipping cream,freeze/thaw stable whipped tipping, low fat & lite natural cheddarcheese, low fat Swiss style yoghurt, aerated frozen desserts, andnovelty bars, hard pack ice cream, label friendly, improved economics &indulgence of hard pack ice cream, low fat ice cream: soft serve,barbecue sauce, cheese dip sauce, cottage cheese dressing, dry mixAlfredo sauce, mix cheese sauce, dry mix tomato sauce and others.

The bran composition of the present invention can also be used inbeverages, in particular alcoholic beverages such as beer.

The invention therefore also provides a kit of parts comprising:

(a) a cereal bran comprising oat bran, rye bran or a mixture thereof;(b) an enzyme comprising a cellulase enzyme, a glucanase and/or xylanaseenzyme or a mixture thereof;(c) instructions for use in a method according to the invention; and(d) optionally other ingredients for a food product.

EXAMPLES Materials and Methods Enzymes

The enzyme composition used in these Examples is a mixture of enzymes,comprising:

(a) a bacterial xylanase component which is found in the commercialproduct available from DuPont Nutrition Biosciences ApS under the tradename Powerbake 930 (referred to in WO 2010/081869); and(b) a glucanase and cellulase components derived from a fermentation ofTrichoderma reesei as referred to in PCT/EP2015/080439, unpublished atthe filing date of the present application and available from DuPontNutrition Biosciences ApS under the trade name Laminex BG2.

This specific composition is referred to in these Examples as “TSE1732”.

The xylanase component (a) was present in an amount of 5400 units ofxylanase activity (XU) per g enzyme composition, the units beingmeasured in accordance with the xylanase activity assay referred toabove.

The glucanase/cellulase component (b) was present in an amount of 2900units of cellulase activity per g enzyme composition, the units beingmeasured in accordance with the CMC-DNS activity assay referred toabove.

Some of the Examples also use a lipase referred to therein as “PB4070”,which is a glycolipase available from DuPont Nutrition Biosciences ApS.This is an enzyme from Fusarium heterosporum and is disclosed inWO2005/087918.

Bran

Rye bran, batch 5011582, from Lantmännen SwedenOat bran, batch 2014-00075, from Lantmännen Sweden

Volume Measurements

Volume measurements of the final bread were carried out using acalibrated rapeseed displacement volume meter from National MFG, USA.Volume determination is based on AACC International Method 10-05.01.After weighing of the bread the specific volume of the bread is reportedas volume in cubic centimetres (cm³) per g of bread.

Hardness Measurements

Hardness of bread slices was determined from a texture profile analysis(TPA) using a Texture analyser TAXTplus from Stable Microsystems.

Example 1—Enzyme Treatment of Bran Samples

The experimental setup and results of the enzyme treatment of bran isshown in Table 1. A slurry of bran, water and the enzyme composition TSE1732 was heated in a heating vessel to 50° C. and kept at thistemperature for 4 hours. Then the temperature was brought to 95° C. for15 minutes to inactivate the enzymes and then cooled to roomtemperature. The treated bran composition of the invention was stored at−20° C. Solubilisation was determined according to the bransolubilisation method described previously.

TABLE 1 Solubi- Bran Water, TSE 1732, TSE 1732, bran, lisation, SampleType g ppm g g % 1 Oat bran 14400 2000 9.6 4800 54 2 Oat bran 2400 0 0800 — 3 Rye bran 22500 3125 23.4 7500 51 4 Rye bran 2400 0 0 800 39

Example 2—Baking Trials in 100% Wheat Flour

The samples generated in Table 1 were tested in standard toast breadusing 100% wheat flour. Water addition was adjusted to obtain the samedough viscosity in each trial. All the bran samples were dosed in at 6%(bran dry solids) meaning that dry solids bran will constitute 6% of thetotal flour composition.

The setup and results are shown in Table 2, wherein:

“Heated oat bran” means Sample 2 from Table 1;“Enzyme treated oat bran” means Sample 1 from Table 1;“Heated rye bran” means Sample 4 from Table 1; and“Enzyme treated rye bran” means Sample 3 from Table 1.

TABLE 2 Trial 1 2 3 4 5 6 7 8 9 Oat bran, % — 6 — — — — — — — Heated oatbran, % — — 6   — — — — — — Enzyme treated oat — — — 6   — — — — — bran,% Enzyme treated oat — — — — 6   — — bran, % + 40 ppm PB4070 Rye bran, %— — — — — 6 — — — Heated rye bran, % — — — — — — 6   — — Enzyme treatedrye — — — — — — — 6   — bran, % Enzyme treated rye — — — — — — — — 6bran, % + 40 ppm PB4070 Specific volume, cm³/g 4.86 4.59 4.82 5.01 5.124.57 4.76 4.59 4.83 Hardness (day 1), N 4.67 4.62 3.76 3.80 3.49 4.484.15 4.38 3.98 Hardness (day 7), N 10.94 9.38 8.56 8.50 8.33 10.26 8.467.80 7.25

A picture showing the crumb of the final breads is shown in FIG. 1.

From these experiments it can be concluded as follows:

Oat Bran:

Enzyme treatment of the oat bran according to the invention had apositive effect on the specific volume of bread incorporating the oatbran, compared with bread containing untreated oat bran. Breadincorporating the enzyme-treated bran had almost the same volume as thatproduced from 100% wheat flour. In addition, bread formed from brantreated with the enzyme composition and lipase had higher specificvolume.

Furthermore, it can be seen that bread containing the treated oat branaccording to the invention is softer on day 1 and day 7, compared withbread containing untreated oat bran.

Rye Bran:

It can be seen that bread containing the treated rye bran according tothe invention is softer on day 1 and day 7, compared with breadcontaining untreated rye bran.

Example 3—Baking Trials in 70% Rye Flour and 30% Wheat Flour, OpenBreads

The samples generated in Table 1 were tested in open Mischbrot bakingtrials using 70% rye flour and 30% wheat flour. Water addition wasadjusted to obtain the same dough viscosity in each trial. All the bransamples were dosed in at 6% (bran dry solids) meaning that dry solidsbran will constitute 6% of the total flour.

The setup and results are shown in Table 3, wherein the terms mean thesame as in Table 2.

TABLE 3 Trial 1 2 3 4 5 6 7 8 9 Oat bran, % — 6 — — — — — — — Heated oatbran, % — — 6 — — — — — — Enzyme treated oat — — — 6 — — — — — bran, %Enzyme treated oat — — — — 6 — — — — bran, % + 40 ppm PB4070 Rye bran, %— — — — — 6 — — — Heated rye bran, % — — — — — — 6 — — Enzyme treatedrye — — — — — — — 6 — bran, % Enzyme treated rye — — — — — — — — 6 bran,% + 40 ppm PB4070 Specific volume, cm³/g 2.06 2.12 2.14 2.25 2.71 2.032.01 2.12 1.87 Hardness (day 7), N 24.63 18.26 15.59 13.07 6.44 18.6218.52 12.76 15.32

FIG. 2 shows the crumbs of the final breads.

From these experiments it can be concluded as follows:

Oat Bran:

Enzyme treatment of the oat bran according to the invention had apositive effect on the specific volume of bread incorporating the oatbran, both compared with bread containing untreated oat bran andcompared with bread produced from flour to which oat bran had not beenadded. In addition, bread formed from bran treated with the enzymecomposition and lipase had higher specific volume.

Furthermore, it can be seen that bread containing the treated oat branaccording to the invention is softer on day 7, compared with breadcontaining untreated oat bran and compared with bread produced fromflour to which oat bran had not been added.

Rye Bran:

Enzyme treatment of the rye bran according to the invention had apositive effect on the specific volume of bread incorporating the ryebran, both compared with bread containing untreated rye bran andcompared with bread produced from flour to which rye bran had not beenadded. It can also be seen that bread containing the treated rye branaccording to the invention is softer on and day 7, compared with breadcontaining untreated rye bran.

Example 4—Baking Trial in 70% Rye Flour and 30% Wheat Flour, Baked inTins

The samples generated in Table 1 were tested in tin baked Mischbrotbaking trials using 70% rye flour and 30% wheat flour. Water additionwas adjusted to obtain the same dough viscosity in each trial. All thebran samples were dosed in at 6% (bran dry solids) meaning that drysolids bran will constitute 6% of the total flour.

The setup and results are shown in Table 4, the terms having the samemeaning as in Table 2.

TABLE 4 Trial 1 2 3 4 Oat bran, % — 6 — — Enzyme treated oat bran, % — —6 — Enzyme treated oat bran, — — — 6 % + 40 ppm PB4070 Specific volume,cm³/g 1.9 1.96 1.98 1.99 Hardness (day 1), N 11.28 6.59 9.90 7.94Hardness (day 7), N 23.14 15.75 14.50 15.77

From these experiments it can be concluded that enzyme treatment of theoat bran according to the invention had a positive effect on thespecific volume of bread incorporating the oat bran, both compared withbread containing untreated oat bran and compared with bread producedfrom flour not containing oat bran. Furthermore, it can be seen thatbread containing the treated oat bran according to the invention issofter compared with bread produced from not containing oat bran.

Example 5—Baking Trial in 100% Rye Flour

The samples generated in Table 1 were tested in baking trials using 100%rye flour. Water addition was adjusted to obtain the same doughviscosity in each trial. All the bran samples were dosed in at 6% (brandry solids) meaning that dry solids bran will constitute 6% of the totalflour.

The setup and results are shown in Table 5, the terms having the samemeaning as in Table 2.

TABLE 5 Trial 1 2 3 4 Oat bran, % — 6 — — Enzyme treated oat bran, % — —6 — Enzyme treated oat bran, — — — 6 % + 40 ppm PB4070 Specific volume,cm³/g 1.5 1.49 1.54 1.55 Hardness (day 3), N 25.37 21.70 16.14 14.42Hardness (day 7), N 28.78 27.03 18.04 17.84

From these experiments it can be concluded that enzyme treatment of theoat bran according to the invention had a positive effect on thespecific volume of bread from 100% rye flour incorporating the oat bran,both compared with bread containing untreated oat bran and compared withbread produced from 100% rye flour. Furthermore, it can be seen thatbread containing the treated oat bran according to the invention issofter, both compared with bread containing untreated oat bran andcompared with bread produced from 100% rye flour.

Various modifications and variations of the described methods and systemof the present invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the present invention.Although the present invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in foodscience, biochemistry and biotechnology or related fields are intendedto be within the scope of the following claims.

1. A composition comprising: (a) a cereal bran comprising oat bran, ryebran or a mixture thereof; and (b) an enzyme composition comprising acellulase enzyme, a glucanase enzyme, a xylanase enzyme or a mixturethereof.
 2. A composition according to any preceding claim, wherein theenzyme composition causes the cereal bran to be at least partiallymodified.
 3. A composition according to claim 1, wherein the cereal branis oat bran.
 4. A composition according to claim 1, wherein the cerealbran is rye bran.
 5. A composition according to claim 4, wherein theenzyme composition comprises a mixture of a cellulase enzyme, aglucanase enzyme and/or a xylanase enzyme.
 6. A composition according toclaim 5, wherein the cellulase enzyme is present in an amount of 0.1 mgto 100 mg per 100 g bran.
 7. A composition according to claim 6, whereinthe cellulase enzyme is present in an amount of 30 to 3000 units ofcellulase activity per 100 g bran.
 8. A composition according to claim7, wherein the xylanase enzyme is present in an amount of 0.006 mg to0.6 mg per 100 g bran.
 9. A composition according to claim 8, whereinthe xylanase enzyme is present in an amount of 50 to 5000 units ofxylanase activity (XU) per 100 g bran.
 10. A composition according toclaim 9, which is a liquid suspension containing the cereal bran and theenzyme composition.
 11. A composition according to claim 10, which is anaqueous suspension containing the cereal bran and the enzymecomposition.
 12. A composition according to claim 11, wherein the enzymecomposition causes the cereal bran to be at least partially solubilised.13. A composition obtained or obtainable by drying the compositionaccording to claim
 12. 14. A composition according to claim 13, furthercomprising a lipase enzyme.
 15. A method of preparing a compositionaccording to claim 14, comprising contacting the cereal bran with theenzyme and, optionally, other components of the composition.
 16. Amethod according to claim 15, further comprising the addition of waterto form an aqueous suspension of the cereal bran and the enzymecomposition.
 17. A method according to claim 16, further comprisingheating the aqueous suspension.
 18. A method according to claim 17,wherein the aqueous suspension is heated to a temperature of 30° C. to100° C.
 19. A method according to claim 18, wherein the aqueoussuspension is heated for a time of 10 minutes to 48 hours.
 20. A flourcomposition comprising: (a) a flour; and (b) a composition according toclaim
 14. 21. A flour composition according to claim 20, wherein theflour comprises wheat flour.
 22. A flour composition according to claim20, wherein the flour comprises rye flour.
 23. A flour compositionaccording to claim 20, wherein the flour comprises a mixture of wheatflour and rye flour.
 24. A food product containing a compositionaccording to claim
 1. 25. A dough composition comprising: (a) the flourcomposition according to claim 20; and (b) water.
 26. A baked productobtainable by baking the dough composition according to claim
 25. 27. Abaked product according to claim 26, said baked product comprisingbread.
 28. A method of solubilising a cereal bran, said methodcomprising treating a liquid suspension of said cereal bran with anenzyme; wherein: (a) said cereal bran comprises oat bran, rye bran or amixture thereof; and (b) said enzyme comprises a cellulase enzyme, aglucanase enzyme and/or xylanase enzyme or a mixture thereof.
 29. Amethod according to claim 28, wherein less than 30% by weight of theliquid suspension is starch or components containing starch.
 30. Amethod according to claim 28, wherein less than 20% by weight of theliquid suspension is starch or components containing starch. 31.Solubilised cereal bran obtained or obtainable according to the methodof claim
 30. 32. A food ingredient obtained or obtainable by drying thesolubilised cereal bran of claim
 31. 33. Use of a solubilised cerealbran according to claim 28 in the preparation of a food product.
 34. Useaccording to claim 33, wherein the food product is a dough or a bakedproduct prepared from dough.
 35. A kit of parts comprising: (a) a cerealbran comprising oat bran, rye bran or a mixture thereof; (b) an enzymecomprising a cellulase enzyme, a glucanase and/or xylanase enzyme or amixture thereof; (c) instructions for use in a method according to claim15; and (d) optionally other ingredients for a food product.
 36. Use ofan enzyme to improve the volume of bread containing a cereal bran,wherein: (a) said cereal bran comprises oat bran, rye bran or a mixturethereof; and (b) said enzyme comprises a cellulase enzyme, a glucanaseand/or xylanase enzyme or a mixture thereof.
 37. Use of an enzyme toimprove the softness of bread containing a cereal bran, wherein: (a)said cereal bran comprises oat bran, rye bran or a mixture thereof; and(b) said enzyme comprises a cellulase enzyme, a glucanase enzyme and/orxylanase enzyme or a mixture thereof.